Studies in this competitive renewal will test the hypothesis that the propensity for silencing of tumor suppressor genes in the respiratory epithelium of chronic smokers by promoter hypermethylation is influenced by sequence variations that modify the activity of genes and microRNAs that directly or indirectly influence de novo methylation and chromatin remodeling. This hypothesis will be tested through four specific aims. Aim 1 will discover and validate gene sequence variations, copy number variants, and haplotypes that are determinants of gene promoter hypermethylation using a whole genome-wide association study (GWAS). A GWAS using the Illumina 610K BeadChip will be performed on 1,500 non-Hispanic white members of the Lovelace Smokers Cohort to identify SNPs significantly associated with extent of methylation of an 8-gene panel in sputum. SNPs across the genome; within microRNA coding and 3' untranslated regions of genes will be identified and validated for their association with methylation in 1,000 members of the PluSS Cohort from the Pittsburgh Lung SPORE and then assessed for association with lung cancer through multi-center case- control studies. Aim 2 will focus on identifying sequence variation in the promoter region of precursor microRNAs and their target genes involved in cytosine methylation and histone modification and evaluate their association with gene methylation and lung cancer. Aim 3 will translate findings from the population-based studies back to the laboratory bench to address how the identified gene haplotypes and SNPs associated with methylation in sputum affect the pathways in which they reside and influence the propensity for methylation. Aim 4 will address whether integrating validated SNPs and haplotypes identified through GWAS and associated with methylation and lung cancer can improve the positive and negative predictive value of gene methylation detection in sputum for early lung cancer diagnosis. A nested, case-control study within ACRIN, a prospective cohort of people at high risk for cancer within the National Lung Screening Trial will be conducted through the Johns Hopkins and Colorado Lung SPOREs. Gene methylation assays in sputum will be conducted through our SPORE project with Johns Hopkins and genotyping of validated SNPs and haplotypes will be done through this grant. These studies are highly relevant to the mission of the NCI to reduce the suffering and health burden of cancer: they will uncover genes and microRNAs whose altered regulation due to inherited sequence variations are critical for the silencing of genes by promoter hypermethylation, a key-step in the development of lung cancer and likely an intermediate marker for risk. Therefore, these studies will identify biomarkers for risk assessment and early detection of lung cancer, the leading cause of cancer-related death in the U.S.